The Aryl hydrocarbon Receptor (AhR) is a mediator of xenobiotic toxicity, best recognized for conveying the deleterious human health effects following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related environmental contaminants. Mechanistically, the AhR is known to function as a ligand-activated transcription factor that binds to a canonical xenobiotic response element (XRE) in association with its heterodimerization partner, the AhR nuclear translocator (Arnt) protein. However, within the repertoire of AhR target genes identified in recent years, many lack a clearly defined XRE, highlighting the growing realization that AhR-mediated gene expression appears to involve additional mechanisms distinct from the well- characterized process involving the XRE. We recently identified a non-consensus XRE (NC-XRE) that recruits a novel AhR-containing protein-DNA complex. DNA binding and functional studies confirmed that NC-XRE binding by the AhR is independent of the Arnt protein, and instead occurs in partnership with the Kruppel-like factor 6 (KLF6) tumor suppressor. Mass spectrometry-based proteomics identified Carbamoyl-Phosphate Synthase 1 (CPS1) as an additional component of the TCDD induced AhR-KLF6 NC-XRE-bound complex. Preliminary evidence indicates that TCDD treatment promotes reversible site-specific homocitrullination (carbamoylation of lysine residues) of lysine 34 on histone H1, supporting the hypothesis that CPS1 recruitment to the NC-XRE complex catalyzes a hitherto unknown epigenetic modification responsible for chromatin remodeling and transcriptional regulation. Our evidence also reveals that AhR-dependent transcriptional activation of the peptidylarginine deiminase type-2 (PADI2) gene requires CPS1 recruitment. Since the PADI2 protein modifies arginine 26 on histone H3 to facilitate transcriptional activation, the data are consistent with the AhR catalyzing a distinct epigenetic signature through the direct and indirect action of specific chromatin modifying enzymes. In keeping with our long-term objective to understand the pathophysiology of AhR activity, this proposal will examine three specific aims designed to characterize the epigenetic modifications initiated by the recruitment of CPS1 to chromatin. Specific aim 1 describes a detailed characterization of the CPS1 interaction with the novel AhR-KLF6 complex. Specific Aim 2 will test the hypothesis that AhR-mediated recruitment of CPS1 introduces epigenetic modifications that alter chromatin into an open, transcriptionally permissive conformation. Specific Aim 3 will use ChIP-sequencing and whole transcriptome shotgun sequencing to provide a global genomic assessment of the functional CPS1-dependent AhR-KLF6 binding sites. Identification of this novel AhR complex represents a paradigm shift in our understanding of AhR biology, with discovery of the homocitrulline post-translational modification embodying a significant and innovative insight into epigenetic transcriptional control.